In order for animals to mount an immune response to a protein antigen, the antigen has to be first processed to small peptide fragments and presented together with MHC Class II molecules on the surface of an antigen presenting cell. The molecular basis of this event has not been completely elucidated. I propose to use a panel of macrophage variants, which have defects in antigen presentation, to study the mechanisms of antigen processing and presentation. The in vivo variants will be selected from a murine splenic macrophage cell line BAC1 (H-2d/f) by a unique T cell selection strategy. Variants are selected to have defects in their interaction with helper T cells. They have either structural alterations of their MHC Class II I-Ed molecules or modifications of their antigen processing compartments. Analyzing the panel of structural mutants at molecular level will enable us to identify the structure elements on the I- Ed molecule which are important for T cell recognition and antigen binding. Analyzing the panel of processing mutants at a subcellular level will help us gain insight into the antigen processing event.